Antibiotic A26201-1 and antibiotic A26201-2 produced by a novel strain of actinoplanes

ABSTRACT

Antibiotic A26201-1 and antibiotic A26201-2 are produced by the novel microorganism, Actinoplanes species A26201 (ATCC 39573), under aqueous aerobic fermentation conditions. The antibiotics are useful for inhibiting bacteria, particularly gram positive bacteria, and are also useful for promoting the growth of monogastric and ruminant animals.

BACKGROUND OF THE INVENTION

Substances active against microorganisms have many beneficial uses.These uses are in fields such as human health care, veterinary science,and animal husbandry. Antimicrobial agents can have many desirableeffects such as preventing or curing disease and promoting the growth ofanimals.

New antimicrobial agents are needed for several reasons; these includeintolerance of the subject to be treated to known antimicrobials, andthe development of strains resistant to known antimicrobials. Thereforecharacterization of any previously known microorganisms which producenew antimicrobial agents is highly desirable.

SUMMARY OF THE INVENTION

The present invention is directed to novel biologically activesubstances produced by the fermentation of the novel microorganismActinoplanes species A26201 and the process of production thereof.Although this organism is a member of the genus Actinoplanes, it cannotbe placed into any of the previously known species and thereforerepresents a previously unknown species of Actinoplanes. Processes usingmutant organisms derived from the species disclosed herein areconsidered to be within the scope of this invention. Mutant organisms ofActinoplanes species A26201 may be obtained by chemical or physicaltechniques, or by other techniques appreciated in the art. A subcultureof Actinoplanes species A26201 has been made part of the permanentcollection of the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., where it is assigned the accession No. 39573.

The novel biologically active substances of this invention which areproduced by the fermentation of Actinoplanes species A26201 haveantimicrobial and/or growth promotion activities. For simplicity ofdiscussion, these substances will be referred to generally as"antibiotics." The term "crude broth material" refers to an unpurifiedmixture of antibiotics consisting of a dried or aqueous aliquot ofculture medium having antibiotic and/or growth promotion activity afterfermentation by Actinoplanes species A26201. The term "antibioticA26201" refers to a partially purified mixture of antibiotics derivedfrom the crude broth material. A step-gradient elution system usingacetonitrile in water on a reverse phase preparative liquidchromatography system was used to obtain this partially purifiedmaterial which includes the two novel active components. The terms"antibiotic A26201-1" and "antibiotic A26201-2" refer to two distinct,purified, active components contained in the crude broth material and inantibiotic A26201. The term "appropriate microorganism" refers to amicroorganism capable of producing antibiotic A26201-1 and/or antibioticA26201-2 such as Actinoplanes species A26201 or any other microorganismsuch as mutants of Actinoplanes species A26201 which is capable ofproducing at least one of said antibiotics.

The antibiotics of this invention are differentiated from knownsubstances by their chemical and physical properties as well as theirrange of antibiotic activity. The term "inhibition" or "inhibiting"refers to antimicrobial activity such as the suppression, control, kill,stasis, or destruction of microorganisms, or any interference with thegrowth of microorganisms which results in a slower growth rate. The term"effective amount" refers to that amount of biologically activesubstance sufficient to result in inhibition of microorganisms.

The present invention is also directed to a method of producing thenovel antibiotics of this invention which comprises growing Actinoplanesspecies A26201 in a suitable nutrient medium and recovering theantibiotics therefrom. The antibiotics of this invention are useful asantimicrobial agents and/or as growth promoting agents in monogastricand ruminant animals. The term "animals" refers to those animals inwhich it is desirable to increase the growth rate and/or feed conversionefficiency; the term "monogastric animals" refers to those animalswithout a developed rumen function in which it is desirable to increasethe growth rate and/or the feed conversion efficiency such as swine orpoultry; the term "ruminant animals" refers to those animals with adeveloped rumen function in which it is desirable to increase the growthrate and/or feed conversion efficiency such as cattle or sheep; the term"growth promoting amount" refers to that amount of antibiotic orantibiotics sufficient to increase the growth rate and/or feedconversion efficiency of the treated animals without resulting in anysignificant adverse side effects; the term "standard animal feed" refersto customary animal feed for monogastric and/or ruminant animals ascommonly known in the art.

DESCRIPTION OF THE DRAWINGS

FIG. 1--Reverse phase high performance liquid chromatograph illustratingthe separation of the fractions containing antibiotic A26201-1 andantibiotic A26201-2. Inj. refers to where the sample was injected.

FIG. 2--Ultraviolet absorption spectrum of antibiotic A26201-1.

FIG. 3--Ultraviolet absorption spectrum of antibiotic A26201-2. Theordinate limit for the dashed line representing 0.1N NaOH in methylalcohol (MeOH) was changed to 2.0 to accommodate the new λ maximum at218.6 nanometers.

FIG. 4--Infrared absorption spectrum of antibiotic A26201-1.

FIG. 5--Infrared absorption spectrum of antibiotic A26201-2.

FIG. 6--Proton nuclear magnetic resonance spectrum of antibioticA26201-1. The spectrum contains a dioxane spike which was included as aninternal control.

FIG. 7--Proton nuclear magnetic resonance spectrum of antibioticA26201-2.

DETAILED DESCRIPTION OF THE INVENTION

The genus Actinoplanes is characterized in "Bergey's Manual ofDeterminative Bacteriology," 8th edition, Williams & Wilkins, 1974, ashaving sporangia which are 3 to 20 by 6 to 30 micrometers (μm) in size.The sporangia can be spherical, subspherical, cylindrical with roundedends, or very irregular. The spores are globose to subglobose, 1 to 1.5μm in diameter, occur in coils, nearly straight chains, or areirregularly arranged in sporangia. The spores are also motile by a tuftof polar flagella 2 to 6 μm in length. The hyphae are 0.2 to 2.6 μm indiameter, branched, irregularly coiled, twisted or straight with fewsepta. Vertical pallisade hyphae are formed on certain agars; aerialmycelia are scanty, except in A. armeniacus. Most species arebrilliantly colored on peptone Czapek and certain other agars; colorsare orange, red, yellow, violet and purple. Some strains form diffusiblepigments which color the agar blue, red, yellow, brownish or greenish.No organic growth factors are required H₂ S is produced by some species.All members of the genus are strict aerobes. Temperature range forgrowth is 18° C. to 35° C. The organisms occur on a wide variety ofplant material, less often on parts of dead animals such as hair, hoofs,snake skin. The Guanine+Cytosine content of the DNA (of two speciesstudied) ranges from 72.1 to 72.6 mole %.

The novel microorganism, Actinoplanes species A26201, was isolated froma soil sample. This species grows well on a variety of nutrient mediaresulting in characteristic macroscopic morphology. For example, after 5to 7 days incubation on oatmeal agar, the colonies are about 5 to 6millimeters (mm) in diameter and have an undulate surface. A sparsemycelium and orange pigment are also produced. On some other media,abundant aerial mycelia are produced.

When grown on most agar media, Actinoplanes species A26201 producessporangia; these sporangia are frequently seen on the central portion ofindividual colonies. The sporangia are spherical to oval in shape, haveregular contours and a diameter ranging from about 15 to 25 μm.Sporangiospores are straight, about 15 μm long with a diameter of about2 μm. The spores are highly motile and are spherical to oval with adiameter of about 1.5 to 2 μm.

After fermentation of Actinoplanes species A26201, antibiotic A26201-1and antibiotic A26201-2 are found in the fermentation broth. The crudebroth material can be partially purified by standard purificationprocedures such as various chromatographic techniques. The preferredmethod is preparative reverse phase liquid chromatography using a stepgradient which yields antibiotic A26201. Antibiotic A26201 can then beresolved into antibiotic A26201-1 and antibiotic A26201-2 by separationtechniques such as a semipreparative reverse phase high performanceliquid chromatography system. The individual antibiotics are thenfurther purified by removing any buffers and solvents required in theseparation procedure. The purified antibiotics can then be lyophilyzed.

Antibiotic A26201 contains amino acids and inhibits cell wall synthesis.These characteristics are shared by known antibiotics produced by otherActinoplanes species, see U.S. Pat. Nos. 4,239,751; 4,303,646;4,375,513; and J. Antibiotics, 29, 501-506, 511-515 (1976). Theantibiotics produced by Actinoplanes species A26201 most closelyresemble gardimycin which is produced by either Actinoplanesgarbadinensis or Actinoplanes liguriae. However, antibiotic A26201-1,antibiotic A26201-2, and gardimycin can be differentiated by theirrespective physical and chemical characteristics. In addition,Actinoplanes species A26201, Actinoplanes garbadinensis, andActinoplanes liguriae can be differentiated by their respectivecultural, biochemical, and physiological characteristics, as well astheir ability to use various carbon sources.

In the preparation of the novel antibiotics of this invention,Actinoplanes species A26201 is cultivated under aerobic conditions in anutrient medium suitable for its growth until the antibiotics areproduced by said microorganism in said culture medium. For example, themaintenance, seed, and fermentation media described herein areinoculated and incubated for about 2 to 14 days, 5 to 7 days beingpreferred, at a temperature of about 20° C. to 35° C., 28° C. to 30° C.being preferred. Incubation with agitation is preferred with aqueousmedia. The pH of the media described herein prior to inoculation isabout 6 to 8, with 6.8 to 7.3 being preferred.

Actinoplanes species A26201 is capable of using at least one of severalconventional nitrogen sources in a concentration from about 0.1% to 10%of medium such as casamino acids, HY-SOY®, N-Z amine, Brain Heartinfusion, Trypticase Soy, Peptone, and Casitone. The carbohydrate sourcecan be at least one of several common carbohydrates such as glucose,starch, mannose, fructose, glycerol, lactose, sucrose and maltose, withfructose and sucrose being preferred at a concentration from about 0.1%to 10% of medium. Essential trace minerals and trace elements can alsobe added to the medium. Frequently, such trace minerals and elementsoccur as impurities in other constituents of the medium in amountssufficient to meet the growth requirements of the organism.

Addition of at least one amino acid such as tryptophan or valine to themedia further stimulates fermentation activity.

To determine antibiotic activity an assay using a susceptiblemicroorganism such as Clostridium perfringens or Sarcina lutea can beemployed.

The antibiotics of the present invention can be used in a wide varietyof applications in which inhibition of microorganisms is desired. Theantibiotics are active against pathogenic and non-pathogenic bacteriawhich may be resistant to widely used known antibiotics. Because of thisactivity, the antibiotics of the present invention can be used astherapeutic agents either alone or in combination withpharmaceutically-acceptable carriers.

The antibiotics of the present invention or combinations containing thesame can also be used as disinfectants, for example, to disinfectobjects and instruments. The novel antibiotics can be used asantibacterial agents, for example, by contacting bacterial pests ortheir habitat with effective amounts sufficient to obtain inhibition ofmany organisms. The antibiotics of this invention can be incorporatedinto various products susceptible to microbial degradation in order toprevent such degradation of the products by the microorganisms.

The novel antibiotics can also be used as growth promoting agents inanimals. In monogastric or ruminant animals, the antibiotics can beadministered to said animals by common means appreciated by one skilledin the art (for example, see the methods taught in U.S. Pat. Nos.4,185,091; 4,209,518; 4,333,923; incorporated herein by reference). Inmonogastric animals, the antibiotics can be administered in combinationswith standard animal feed wherein the concentration of said antibioticor antibiotics is about 5 to 25 parts per million (ppm) of the ultimatefeed composition. The novel antibiotics can also be administered toruminant animals by means of combinations with standard animal feedwherein the concentration of said antibiotic or antibiotics is about 2to about 25 ppm of the ultimate feed composition.

The present invention is further illustrated by the following examples;however, these examples are not to be interpreted as a limitation uponthe scope of the present invention.

EXAMPLE 1

A new species of Actinoplanes, referred to herein as Actinoplanesspecies A26201 (ATCC 39573) has been isolated. This species is capableof producing the active substances, antibiotic A26201-1 and antibioticA26201-2. The cultural characteristics on different media ofActinoplanes species A26201 as well as those of two related species ofActinoplanes (A. garbadinensis and A. liguriae) are shown in Table 1.The cultural characteristics were determined after 5 to 7 daysincubation at 28° to 30° C.

The alpha numeric codes in Table 1 refer to standard color references asdescribed by Maery and Paul (Maery, A. and Paul, M., A Dictionary ofColor, 2nd ed., (1950) McGraw-Hill, New York).

                  TABLE 1                                                         ______________________________________                                        Cultural Characteristics of Some Actinoplanes Species                         Culture Medium                                                                          Cultural Characteristics                                            ______________________________________                                                  Species A26201                                                      Bennett's Agar                                                                          Moderate growth with worty surface, orange                                    surface light orange.                                               Czapek    Moderate growth with raised worty surface,                          Glucose Agar                                                                            orange 10/G/9.                                                      Czapek    Moderate growth with raised worty surface,                          Sucrose Agar                                                                            mycelia present 9/A/2.                                              Glucose   Heavy rounded smooth orange growth,                                 Asparagine                                                                              sparse mycelia, sparse sporangia.                                   Agar                                                                          Potato Agar                                                                             Sparse orange round smooth colonies.                                Nutrient Agar                                                                           Sparse orange growth 10/G/6, few sporangia,                                   amber diffusible pigment 13/E/4.                                    Peptone   Moderate orange worty growth 10/G/9,                                Glucose Agar                                                                            Brown Diffusible pigment 13/E/8.                                    Potato Plug                                                                             Poor thin growth with wrinkled surface, light                                 orange 10/A/3.                                                      Yeast Extract                                                                           Heavy thick wrinkled growth, orange,                                Malt Agar sporangia, mycelia 9/A/2.                                           Oatmeal   Sparse undulate growth, orange, widely                              Agar 20%  spread sparse mycelia 9/A/2.                                        Inorganic Moderate thin layer growth, light orange                            Salts Agar                                                                              10/E/4.                                                             Glycerol  Round raised smooth growth, orange,                                 Asparagine                                                                              pink aerial mycelia 9/A/2.                                          Agar                                                                          Peptone-yeast                                                                           Very light growth, orange 10/G/9. Purple                            Extract   soluble pigment 56/E/4.                                             Iron Agar                                                                     Tyrosine Agar                                                                           Heavy growth, raised smooth round colonies                                    orange 10/G/9, sparse mycelia 9/A/2.                                Skim Milk Moderate smooth round growth, orange 10/G/9.                        Agar      Sparse sporangia 9/A/2, brown soluble pigment                                 13/E/8.                                                             Agar      no growth.                                                                    A. garbadinensis                                                    Bennett's Agar                                                                          Abundant growth, wrinkled surface, light                                      orange.                                                             Czapek    Poor growth, smooth surface, light orange.                          Glucose Agar                                                                            Traces of rudimentary aerial mycelium.                              Czapek    Poor growth, crusty surface, light orange.                          Sucrose Agar                                                                  Glucose   Abundant growth, crusty surface, deep                               Asparagine                                                                              orange.                                                             Agar                                                                          Potato Agar                                                                             Abundant growth, wrinkled surface, orange                                     to light brown. Traces of rudimentary                                         aerial mycellium.                                                   Nutrient Agar                                                                           Moderate growth, crusty surface, orange                                       to light brown.                                                     Peptone   Very scanty growth, smooth surface,                                 Glucose Agar                                                                            hyaline.                                                            Potato Plug                                                                             Scanty growth, wrinkled surface,                                              orange.                                                             Yeast Extract                                                                           Abundant growth, wrinkled surface,                                  Malt Agar amber.                                                              Oatmeal   Moderate growth, smooth, opaque, cream                              Agar 20%  to orange at edges.                                                 Inorganic Moderate growth, smooth surface, deep                               Salts Agar                                                                              orange.                                                             Glycerol  Moderate growth, smooth surface,                                    Asparagine                                                                              orange.                                                             Agar                                                                          Peptone-yeast                                                                           Scanty growth, rough surface, dark                                  Extract   brown with brown pigment.                                           Iron Agar                                                                     Tyrosine Agar                                                                           Abundant growth, crusty surface,                                              coffee-colored. Faintly brown                                                 pigment.                                                            Skim Milk Abundant growth, wrinkled surface,                                  Agar      deep orange.                                                        Agar      Very scanty growth, thin and smooth,                                          hyaline.                                                                      A. liguriae                                                         Bennett's Agar                                                                          Abundant growth with crusty surface.                                Czapek    Abundant growth with smooth and thin surface,                       Glucose Agar                                                                            orange. Abundant production of sporangia.                           Czapek    Scarce growth, light orange. Moderate                               Sucrose Agar                                                                            production of sporangia.                                            Glucose   Abundant growth with smooth surface,                                Asparagine                                                                              orange. Some sporangia.                                             Agar                                                                          Potato Agar                                                                             Abundant growth, with smooth surface,                                         amber.                                                              Nutrient Agar                                                                           Abundant growth with smooth surface,                                          orange.                                                             Peptone   Abundant growth, with wrinkled surface,                             Glucose Agar                                                                            deep orange.                                                        Potato Plug                                                                             Scanty growth, wrinkled, light orange.                              Yeast Extract                                                                           Abundant growth, slightly wrinkled,                                 Malt Agar light orange to light amber.                                        Oatmeal   Abundant growth, with smooth and thin                               Agar 20%  surface, light orange. Some sporangia,                                        light yellow soluble pigment.                                       Inorganic Abundant growth with smooth surface,                                Salts Agar                                                                              orange. Some sporangia. Canary                                                yellow soluble pigment.                                             Glycerol  Abundant growth with smooth surface,                                Asparagine                                                                              light orange. Abundant production                                   Agar      of pigment. Canary yellow soluble                                             pigment.                                                            Peptone-yeast                                                                           Moderate growth with smooth surface,                                Extract   orange.                                                             Iron Agar                                                                     Tyrosine Agar                                                                           Abundant growth with smooth surface,                                          rose amber. Good production of                                                sporangia. Rose amber soluble pigment.                              Skim Milk Abundant growth with slightly crusty                                Agar      surface, deep orange. Yellow soluble                                          pigment.                                                            Agar      Very scanty growth thin and smooth.                                           Colorless.                                                          ______________________________________                                    

Table 2 compares the ability of Actinoplanes species A26201,Actinoplanes garbadinensis and Actinoplanes liguriae to use variouscarbohydrate sources. Conventional methods were used for thedetermination. Final concentration of each carbohydrate was 1% of thetotal medium.

                  TABLE 2                                                         ______________________________________                                        Carbohydrate Utilization Pattern                                                        A. species                                                          Carbon Source                                                                           A26201    A. liguriae A. garbadinensis                              ______________________________________                                        C.sub.5 arabinose                                                                       +         +           +                                             xylose    +         +           +                                             C.sub.6 glucose                                                                         +         +           +                                             fructose  +         +           +                                             mannose   +         +           +                                             mannitol  +         -           +                                             inositol  +/-       +           -                                             rhamnose  +         +           +                                             (C.sub.6).sub.2 sucrose                                                                 +         -           +                                             lactose   +/-       -           +                                             (C.sub.6).sub.3 raffinose                                                               +/-       -           -                                             (C.sub.6).sub.n cellulose                                                               -         -           -                                             starch    +         +           +                                             ______________________________________                                         + = utilization                                                               +/- = weak utilization                                                        - = no utilization                                                       

Table 3 compares biochemical and physiological properties ofActinoplanes species A26201, Actinoplanes liguriae and Actinoplanesgarbadinensis. All biochemical and physiological properties were eitherperformed on petri plates according to conventional methods, or an APIstrips (Analytab Products, Planview, N.Y.).

                  TABLE 3                                                         ______________________________________                                        Biochemical and Physiological Properties                                                    A. species                                                                              A.       A.                                           Property      A26201    liguriae garbadinensis                                ______________________________________                                        indole production                                                                           -         -        -                                            starch hydrolysis                                                                           ++        ++       ++                                           urease activity                                                                             +/-       +/-      +                                            gelatin liquefaction                                                                        +/-       -        ++                                           esculin hydrolysis                                                                          ++        -        +/-                                          catalase      +/-       ++       ++                                           hippurate hydrolysis                                                                        -         +/-      -                                            leucine aminopeptidase                                                                      +/-       +        +                                            serine aminopeptidase                                                                       +         +        +                                            pyroglutamic  +         +        +                                            aminopeptidase                                                                arginine aminopeptidase                                                                     +         +        +                                            β-galactosidase                                                                        +         +        +                                            β-glucosidase                                                                          +         +        +                                            alkaline phosphatase                                                                        +         +        +                                            arginine dehydrogenase                                                                      +/-       +        +                                            β-glucosaminidase                                                                      +         +        +                                            indoxale acetate                                                                            +         +        +                                            hydrolysis                                                                    ______________________________________                                         - = negative                                                                  +/- = weak positive                                                           +  = positive                                                                 ++ = strong positive                                                     

From the microscopic and macroscopic morphology, strain A26201 isrecognized as a member of the genus Actinoplanes. Other data indicatethat Actinoplanes species A26201 differs from closely related speciessuch as A. liguriae and A. garbadinensis and represents a new species ofActinoplanes. As seen in Table 3, Actinoplanes species A26201 from A.liguriae with regard to gelatin liquefaction, esculin hydrolysis,hippurate hydrolysis, catalase, leucine aminopeptidase and argininedehydrogenase activities, while Actinoplanes species A26201 differs fromA. garbadinensis with regard to urease activity, gelatin liquefaction,esculin hydrolysis, catalase, leucine aminopeptidase and argininedehydrogenase activities. Table 2 shows that Actinoplanes species A26201differs from Actinoplanes liguriae with regard to utilization ofmannitol, inositol, sucrose, lactose, and raffinose, while Actinoplanesspecies A26201 differs from Actinoplanes garbadinensis with regard toutilization of inositol, lactose and raffinose. All three strains aresufficiently dissimilar to warrant classification as three separatespecies.

EXAMPLE 2

For growth of seed cultures and fermentation cultures, the media E25,modified E25, E10, and CAAYE, shown in Table 4, are suitable. Oatmealagar, also shown in Table 4, is suitable for culture maintenance.

                  TABLE 4                                                         ______________________________________                                        Composition of Media                                                                           Modified        Oatmeal                                      Component                                                                             E25      E25      E10    Agar   CAAYE                                 ______________________________________                                        Glucose 25     g     --       10   g   --       10   g                        Fructose                                                                              --           25   g   --       --       --                            Beef    4      g     4    g   4    g   --       --                            Extract                                                                       Yeast   1.0    g     1.0  g   1.0  g   --       4    g                        Extract                                                                       NaCl    2.5    g     2.5  g   2.5  g   --       --                            Peptone 4      g     4    g   4    g   --       --                            Soybean 10     g     10   g   10   g   --       --                            Meal                                                                          CaCO.sub.3                                                                            5      g     5    g   5    g   --       --                            Casamino                                                                              --           --       --       --       5    g                        Acids                                                                         Difco   --           --       --       200  g   --                            Oatmeal                                                                       Tap Water                                                                             1000   ml    1000 ml  1000 ml  1000 ml  --                            Distilled                                                                             --           --       --       --       1000 ml                       Water                                                                         Agar    --           --       --       20   g   --                            ______________________________________                                    

EXAMPLE 3

A pure culture of Actinoplanes species A26201 was inoculated onto anoatmeal agar slant and incubated at 28° C. to 30° C. for seven days.This maintenance culture was then used to inoculate 100 ml of sterilemodified E25 seed medium in a sterile 500 ml capped culture flask. Theseed culture was then incubated at 28° C. to 30° C. for two days on ashaking apparatus at about 200 revolutions per minute (RPM). Afterfermentation, 10 ml of the seed culture medium was used to inoculate 100ml of sterile modified E25 fermentation medium in a sterile 500 mlcapped culture flask. The resulting fermentation culture was incubatedat 28° C. to 30° C. for six days on a shaking apparatus at about 200RPM's. The pH of the modified E25 seed and fermentation media was about7 prior to inoculation.

The antimicrobial activity of the fermentation broth was determined byuse of a paper-disc agar diffusion system. A portion of the fermentationbroth produced by species A26201 in E25 medium was stored frozen at -20°C. and used as a standard in all assays. One microliter (μl) of theantibiotic standard was arbitrarily designated to contain one unit ofactivity. Various dilutions of the standard were made and 20 μl of eachdilution was pipetted onto paper discs. The discs were then placed ontopreseeded C. perfringens plates. The discs were allowed to dry and theantibiotic allowed to diffuse into the agar. The plates were thenincubated for 16 to 24 hours at 37° C. anaerobically, after which, thesize of the zone was measured. A standard curve was constructed andexperimental data compared to the standard curve to determine theconcentration of antibiotics.

EXAMPLE 4

The active components (i.e., antibiotic A26201-1 and antibioticA26201-2) were isolated from the crude broth material by a step-gradientelution system using acetonitrile (CH₃ CN) in water, on a reverse phasepreparative liquid chromatography system (Waters Prep-500 LC System).The crude broth material, after filtration was adjusted to pH 7 andloaded onto the column then rinsed with 20% acetonitrile in water,followed by elution of the active components in 30% acetonitrile inwater (this preparation of the active components in 30% acetonitrile inwater is antibiotic A26201). No further elution of the active componentsoccurred using 100% acetonitrile. The active components in 30%acetonitrile were separated from each other using 34% CH₃ CN in atriethylamine-phosphate buffer (0.25N H₃ PO₄ adjusted to pH 3.5 usingtriethylamine) on a semi-preparative reverse phase HPLC system (Zorbax®(Dupont) reverse phase (C₁₈) ODS HPLC column (9.4 mm inside diameter×250mm length)) giving two active fractions; one fraction containingantibiotic A26201-1 and the other fraction containing antibioticA26201-2 (see FIG. 1).

The fractions containing antibiotic A26201-1 and antibiotic A26201-2obtained as described above were each respectively treated as follows:The active fraction was slowly loaded onto a reverse phase (Zorbax®,ODS) column and the column rinsed with H₂ O, then washed with lowconcentrations of acetonitrile in H₂ O (5% CH₃ CN, 10% CH₃ CN, 15% CH₃CN, and 20% CH₃ CN) over a period of approximately 1 hour. The purifiedcomponent was eluted with 35% CH₃ CN in H₂ O. After removal of thesolvent (CH₃ CN) by rotary-vaporization, the solution was lyophilized 48hours to give the purified antibiotic, in each case, as a fine whitepowder.

EXAMPLE 5 Characterization of antibiotic A26201-1 and antibioticA26201-2

(A) High Performance Liquid Chromatography (HPLC) Determination

The filtered crude broth material was injected onto a C-18 reverse phaseanalytical HPLC column with an isocratic elution using 30% ultraviolet(uv) grade acetonitrile/70% 32 mM ammonium formate in water. The flowrate was 2 milliliters/minute (min) and uv detection was at 220nanometers. The retention time for antibiotic A26201-1 was 5.11 min andthe retention time for antibiotic A26201-2 was 737 min.

(B) Ultraviolet Absorption Spectra

The ultraviolet absorption spectra of antibiotic A26201-1 and antibioticA26201-2 are shown in FIG. 2 and FIG. 3 respectively. A Perkin-ElmerLamda-3 spectrophotometer (with automatic baseline correction for methylalcohol absorbance) was used for this determination. The maximumabsorptions of the two antibiotics are as follows:

    ______________________________________                                                    Concen-                                                                       tration                                                                              λ max (nanometers)                                                (weight/ Antibiotic Antibiotic                                  Solvent       volume)  A26201-1   A26201-2                                    ______________________________________                                        100% methyl alcohol                                                                         0.1%     205.7, 220.8,                                                                            207.3, 220.5,                                                      291 (minor),                                                                             273 (minor),                                                       218 (inflec-                                                                             282 (minor),                                                       tion)      291.2 (minor),                                                                218 (infec-                                                                   tion)                                       methyl alcohol with                                                                         0.1%     205.3, 220.8,                                                                            204, 220.9,                                 0.1 .sub.--N hydrochloric acid                                                                       292 (minor),                                                                             271.3 (minor),                                                     218 (inflec-                                                                             280.2 (minor),                                                     tion)      289.5 (minor),                                                                212.3 (inflec-                                                                tion)                                       methyl alcohol with                                                                         0.1%     217.4,     218.6,                                      0.1 .sub.--N sodium hydroxide                                                                        292.7 (minor)                                                                            280 (minor),                                                                  289.3 (minor)                               ______________________________________                                    

(C) Infrared Analysis

Antibiotic A26201-1 and antibiotic A26201-2 were subjected to Fouriertransform infrared analysis using a KBr pellet. The infrared spectra ofantibiotic A26201-1 and antibiotic A26201-2 are shown in FIG. 4 and FIG.5, respectively, which reveal similarities between the components andindicate peptide characteristics demonstrated by absorption fromsecondary amide bonds at 1,650 cm⁻¹ and 1,520 cm⁻¹. A sharp peak occursat 2,680 cm⁻¹ and a split peak is noted at 1,000-1,030 cm⁻¹.

(D) Amino Acid and Carbohydrate Analyses

Amino acid analyses of antibiotic A26201-1 and antibiotic A26201-2revealed the presence of the following amino acids in both components:alanine, cysteine, glutamic acid, glycine, isoleucine, leucine, serine,tryptophan, and valine. All amino acid determinations other thantryptophan involved acid hydrolysis followed by high performance liquidchromatography in accordance with standard procedures known in the art.For the cysteine determination, the samples were oxidized with performicacid prior to acid hydrolysis. For the tryptophan determination, themethod of Hugli and Moore, JBC, 247, 2828 (1972), was used whichinvolved an alkaline hydrolysis.

Carbohydrate analyses of both antibiotics ("Phenol Method", Dubois, M,Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F., Anal.Chem., 28, 350-356, (1956)) indicated the presence of only trace amountsof sugars.

(E) Nuclear Magnetic Resonance (NMR) Analysis

Proton NMR analyses were performed on antibiotic A26201-1 and antibioticA26201-2 in D₂ O at 200 ppm the results of which are shown in FIG. 6 andFIG. 7, respectively. Peaks at 2.8-3.3 ppm and 7.2-7.7 ppm can both beattributed to tryptophan. Characteristic peaks are also present atapproximately 1.0 ppm, 1.5 ppm, 2.2 ppm, 2.4 ppm, 3.7 ppm, and 4.6 ppm,respectively.

(F) Elemental Analysis

Elemental analyses of antibiotic A26201-1 and antibiotic A26201-2 wereperformed after drying (48 hours lyophilization at sub-zero temperaturesfollowed by drying in a hot air oven). The results are as follows:

    ______________________________________                                                    Weight %                                                                        Antibiotic                                                                              Antibiotic                                            Element       A26201-1  A26201-2                                              ______________________________________                                        C*            33.2      45.9                                                  H*            7.0       6.8                                                   N*            6.7       12.0                                                  O**           51.7      29.8                                                  S*            1.4       5.5                                                   Cl***         Trace     Trace                                                 ______________________________________                                         *combustion analysis                                                          **by difference (subtraction)                                                 ***neutron =  activation                                                 

From the elemental analysis, simple calculations yield tentativeempirical formulae as follows:

    ______________________________________                                        Antibiotic     Antibiotic                                                     A26201-1       A26201-2                                                       ______________________________________                                        C.sub.55 H.sub.140 O.sub.64 N.sub.10 S                                                       C.sub.45 H.sub.80 O.sub.22 N.sub.10 S.sub.2                    ______________________________________                                    

On the basis of the empirical formulae, estimated minimum molecularweights of the two components were determined to be approximately 2000and 1200, respectively. The relative sulfur analysis is in agreementwith the ratio between cysteine residues of the components.

EXAMPLE 6

The biological activities of the novel antibiotics of this inventionwere determined by various methods as follows:

(A) Antimicrobial Activity

Minimum inhibitory concentrations (MIC's) were determined respectivelyfor the crude broth material, antibiotic A26201, antibiotic A26201-1,antibiotic A26201-2, and gardimycin against a number of differentmicroorganisms. The following techniques were used to determine theMIC's:

Thirty ml aliquots of trypticase soy agar were respectively placed inindividual tubes. The agar was melted and then cooled to about 45° to50° C. A concentration (2-fold serial dilutions of the highestconcentration tested) of test antibiotic was added to each individualtube. Each test mixture was stirred and poured into an individual petriplate so that each petri plate contained a single concentration ofantibiotic. When the agar hardened, the plates were inoculated witheither aerobic or anaerobic test organisms. The plates inoculated withaerobic organisms were incubated at 37° C. for 24 hours and read forbacterial growth. The same plates were again incubated at 30° C. for anadditional 48 hours at which point they were checked for yeast andfungal growth. The plates inoculated with anaerobic organisms wereincubated at 37° C. in an anaerobic chamber and read at 48 hours. In allcases, the MIC's represent the lowest concentration of antibiotic whichdemonstrated suppression of growth. The results are summarized in Table5.

                                      TABLE 5                                     __________________________________________________________________________    Minimum Inhibitory Concentration of Actinoplanes                              Antibiotics Against a Number of Microorganisms                                              MIC (μg/ml)                                                                Crude                                                                         Broth*                                                                             Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                     Organism      Material                                                                           A26201                                                                              A26201-1                                                                            A26201-2                                                                            Gardimycin                               __________________________________________________________________________    Actinomyces viscosus                                                                         1250                                                                               50   5.5   5.5   12.5                                     Clostridium perfringens                                                                       156                                                                                5   2.75  2.75  3.1                                      Clostridium septicum                                                                          312                                                                                5   >11   2.75  3.1                                      Bacteroides fragilis                                                                        >5000                                                                              >50   >11   >22   >50                                      Bacteroides multiacidus                                                                     >5000                                                                              >50   >11   >22   >50                                      Streptococcus faecalis                                                                      >5000                                                                              >50   <11   >22    25                                      Streptococcus mutans                                                                        >5000                                                                              >50   >11   >22   >75                                      Streptococcus bovis                                                                         >5000                                                                              >50   >11   >22   >75                                      Lactobacillus casei                                                                         >5000                                                                              >50   >11   >22   >75                                      Fusobacterium necrophorum                                                                   >5000                                                                              >50   >11   >22   >75                                      Candida albicans                                                                            >5000                                                                              >50   >11   >22   >75                                      Aspergillus niger                                                                           >5000                                                                              >50   >11   >22   >75                                      Mucor miehei  >5000                                                                              >50   >11   >22   >75                                      Pircularia oryzae                                                                           >5000                                                                              >50   >11   >22   >75                                      Bacillus subtilis                                                                            2500                                                                              >50   >11   >22    75                                      Erwinia amylovora                                                                            5000                                                                              >50   >11   >22   >75                                      Staphylococcus aureus                                                                        5000                                                                              >50   >11   >22    75                                      Escherchia coli                                                                             >5000                                                                              >50   >11   >22   >75                                      Salmonella typhimurium                                                                      >5000                                                                              >50   >11   >22   >75                                      Pseudomonas aeruginosa                                                                      >5000                                                                              >50   >11   >22   >75                                      Sarcina lutea   625                                                                               50   2.75  5.5   1.5                                      __________________________________________________________________________     *unaltered aqueous fermentation broth                                    

(B) Monogastric Animal Growth Promotion

The crude broth material from the fermentation of Actinoplanes speciesA26201 was dried onto a portion of standard feed, then the feedcontaining the broth was mixed with a test diet and fed to youngchickens (chicks). The weights of these chicks were compared to theweights of chicks fed a test diet without the crude broth material. Fromthis comparision it was determined that the crude broth materialpromoted the growth of the young chickens.

(C) Ruminant Animal Growth Promotion

The antibiotics of this invention were tested in a 24 hour in vitroruminant growth promotion system.

A fermentation medium useful for carrying out the evaluations describedherein was prepared by the admixture of the following ingredients:

    ______________________________________                                        Mineral solution 1  7.5        ml                                             Mineral solution 2  7.5        ml                                             Micromineral solution                                                                             1.5        ml                                             Resazurin solution 0.1%                                                                           0.1        ml                                             Clarified rumen fluid                                                                             10.0       ml                                             NaHCO.sub.3 (6.33% solution)                                                                      8.0        ml                                             Na.sub.2 S.9H.sub.2 O (2.5% solution)                                                             0.5        ml                                             Distilled water     64.9       ml                                                                 100.0      ml                                             ______________________________________                                    

To each 100 ml of the above preparation, 0.8 g of dry nutrients wasadded. The dry nutrients consisted of 0.3 g of Avicel® PH101-microcrystalline cellulose, 0.3 g of casein, and 0.1 g each ofanhydrous glucose and soluble starch. The pH of the medium was checkedand adjusted to pH 6.8 to 7.2 with CO₂.

The clarified rumen fluid of the fermentation medium was prepared bycollecting rumen fluid from an untreated fistulated cow on a hay dietapproximately 12 hours after feeding. The fluid was strained throughgauze and centrifuged at 5,000 rpm. The supernatant was placed in oneliter amber bottles (about 400 ml/bottle) and autoclaved at about 15pounds of pressure for sterilization.

All of the solutions of the fermentation medium were added as preparedstock solutions of the following compositions (in grams per liter ofwater):

    ______________________________________                                                                grams/liter                                           ______________________________________                                        Mineral Solution 1                                                            K.sub.2 HPO.sub.4         12.5                                                Mineral Solution 2                                                            KH.sub.2 PO.sub.4         12.5                                                MgSO.sub.4.7H.sub.2 O     3.0                                                 NaCl                      12.0                                                CaCl.sub.2.2H.sub.2 O     1.6                                                 Micromineral Solution                                                         FeSO.sub.4.7H.sub.2 O     0.200                                               H.sub.3 BO.sub.3          0.030                                               CoCl.sub.2.6H.sub.2 O     0.020                                               ZnSO.sub.4.7H.sub.2 O     0.010                                               MnCl.sub.2.4H.sub.2 O     0.003                                               Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                           0.003                                               NiCl.sub.2.6H.sub.2 O     0.002                                               CuCl.sub.2.2H.sub.2 O     0.001                                               (pH adjusted to about 2)                                                      Resazurin Solution 0.1%                                                       Resazurin                 1.0                                                 Sodium Bicarbonate Solution 6.33%                                             NaHCO.sub.3               63.3                                                (saturated with, and stored under 100% CO.sub.2)                              Sodium Sulfide Solution 2.5%                                                  Na.sub.2 S.9H.sub.2 O     25.0                                                (stored under nitrogen)                                                       ______________________________________                                    

The in vitro evaluations of antibiotic A26201, antibiotic A26201-1 andantibiotic A26201-2 were carried out in 24 hour batch fermentations inanaerobic digestors having gas and liquid sampling ports and manometersto measure total gas production during the fermentation. Differentconcentrations of antibiotic A26201 (10, 25, and 100 ppm) as well asantibiotic A26201-1 (2.5, 10, and 50 ppm) and antibiotic A26201-2 (2.5ppm) were prepared in 10% methanol solutions and placed in separategroups of digestors.

Fresh rumen fluid (700 ml) from an untreated fistulated cow was added to1300 ml of the fermentation medium previously described, and mixed.After mixing, 10 ml was removed and analyzed as a control, and 200 mlwas placed in each of the digestors. The manometers were attached andnitrogen was bubbled through to remove oxygen. The digestors were thenmaintained at 40° C. while under continuous agitation.

The cultures were sampled at 0, 5 and 24 hours. The 5 hour sample wasused primarily to note effects on nitrogen metabolism. Measurement ofthe change in concentration of protein, amino acids and ammonia in thefermentation after 5 hours was an indication of the extent to which therate of protein degradation and deamination were inhibited by theantibiotics. Concentrations of isoacids, i.e., iso-butyric, iso-valericand valeric acids were determined at 24 hours and used as a measure ofinhibition of deamination since the major source of these acids is thedeamination of the amino acids valine, leucine and proline,respectively.

The 24 hours sample was used to measure volatile fatty acid production.The mole ratio of acetate to propionate (A/P) was used to determine ifthe antibiotics increased the molar proportion of propionate in totalvolatile fatty acid concentrations. The gas composition was alsodetermined at 24 hours and the amount of methane produced was noted. Therate at which gas was produced was determined by reading the manometersat 3, 4, and 5 hours to monitor microbial metabolism. The results of thein vitro fermentations are shown in Table 6. The data generally showthat antibiotic A26201, antibiotic A26201-1, and antibiotic A26201-2improved rumen fermentation efficiency as evidenced by a stimulation ofpropionate production (the main precursor for gluconeogenesis) andinhibition of the less energy efficient acids. The data also showreduced deamination as evidenced by increased amino-N and reducedvalerate and other isoacids (for a more complete discussion of rumenmetabolism see Church et al. in "Digestive Physiology and Nutrition ofRuminants", Vol. 2, 1971, pp. 622 and 625 incorporated herein byreference).

                                      TABLE 6                                     __________________________________________________________________________    Results of Antibiotics in an Artificial Rumen                                             % of Control                                                                  Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                                                          Antibiotic                                A26201                                                                              A26201                                                                              A26201                                                                              A26201-1                                                                            A26201-1                                                                            A26201-1                                                                            A26201-2                                  100 ppm                                                                             25 ppm                                                                              10 ppm                                                                              50 ppm                                                                              10 ppm                                                                              2.5 ppm                                                                             2.5 ppm                       __________________________________________________________________________    CH.sub.4 (methane)                                                                        89    102   111   71    85    89    97                            Gas Rate (ml/min)                                                                         86    91     98   --    78    72    64                            Amino-N mg/100 ml                                                                         166   154   148   195   199   132   166                           Ammonia-N mg/100 ml                                                                       55    63     70   59    66    92    52                            Acetate (A) mM                                                                            100   86    100   69    78    95    77                            Propionate (P)                                                                            130   118   118   108   113   107   101                           Butyrate (B)                                                                              99    103   124   69    86    112   84                            Valerate    18    14     36   15    17    72    41                            Total (A + P + B)                                                                         111   100   109   83    91    101   86                            A/P         77    73     85   64    69    88    76                            Total Isoacids*                                                                           49    58     84   41    50    93    57                            __________________________________________________________________________     *Total Isoacids = Isovalerate + Isobutyrate + Valerate                   

What is claimed is:
 1. Antibiotic A26201-1 having the followingcharacteristics:(1) Ultraviolet absorption spectrum (FIG. 2):

    ______________________________________                                                      Concen-                                                                       tration     λ max (nanometers)                                         (weight/    Antibiotic                                          Solvent       volume)     A26201-1                                            ______________________________________                                        100% methyl alcohol                                                                         0.1%        205.7, 220.8,                                                                 291 (minor),                                                                  218 (inflection)                                    methyl alcohol with                                                                         0.1%        205.3, 220.8,                                       0.1 .sub.--N hydrochloric acid                                                                          292 (minor),                                                                  218 (inflection)                                    methyl alcohol with                                                                         0.1%        217.4,                                              0.1 .sub.--N sodium hydroxide                                                                           292.7 (minor)                                       ______________________________________                                    

(2) Infrared spectrum (FIG. 4):The most important absorption bands occurat the following frequencies (cm⁻¹): 1,650, 1,520, 2,680 (sharp peak)1,000-1,030 (split peak) (3) Amino acid analysis:Presence of alanine,cysteine, glutamic acid, glycine, isoleucine, leucine, serine,tryptophan, and valine (4) Proton NMR analysis (FIG. 6):Characteristicpeaks at (parts per million) 2.8-3.3, 7.2-7.7, 1.0, 1.5, 2.2, 2.4, 3.7and 4.6 (5) Elemental analysis:

    ______________________________________                                        Element      Weight Percentage                                                ______________________________________                                        Carbon       33.2                                                             Hydrogen     7.0                                                              Nitrogen     6.7                                                              Oxygen       51.7                                                             Sulphur      1.4                                                              Chlorine     Trace                                                            ______________________________________                                    

(6) An approximate chemical formula based on the elemental analysis ofC₅₅ H₁₄₀ O₆₄ N₁₀ S with an approximate molecular weight of
 2000. 2.Antibiotic A26201-2 having the following characteristics:(1) Ultravioletabsorption spectrum (FIG. 3):

    ______________________________________                                                      Concen-                                                                       tration     λ max (nanometers)                                         (weight/    Antibiotic                                          Solvent       volume)     A26201-2                                            ______________________________________                                        100% methyl alcohol                                                                         0.1%        207.3, 220.5,                                                                 273 (minor),                                                                  282 (minor),                                                                  291.2 (minor),                                                                218 (inflection)                                    methyl alcohol with                                                                         0.1%        204, 220.9,                                         0.1 .sub.--N hydrochloric acid                                                                          271.3 (minor),                                                                280.2 (minor),                                                                289.5 (minor),                                                                212.3 (inflection)                                  methyl alcohol with                                                                         0.1%        218.6,                                              0.1 .sub.--N sodium hydroxide                                                                           280 (minor),                                                                  289.3 (minor)                                       ______________________________________                                    

(2) Infrared spectrum (FIG. 5): The most important absorption bandsoccur at the following frequencies (cm⁻¹): 1,650, 1,520, 2,680 (sharppeak), 1,000-1,030 (split peak) (3) Amino acid analysis:Presence ofalanine, cysteine, glutamic acid, glycine, isoleucine, leucine, serine,tryptophan and valine (4) Proton NMR analysis (FIG. 7):Characteristicpeaks at (parts per million) 2.8-3.3, 7.2-7.7, 1.0, 1.5, 2.2, 2.4, 3.7and 4.6 (5) Elemental analysis:

    ______________________________________                                        Element      Weight Percentage                                                ______________________________________                                        Carbon       45.9                                                             Hydrogen     6.8                                                              Nitrogen     12.0                                                             Oxygen       29.8                                                             Sulphur      5.5                                                              Chlorine     Trace                                                            ______________________________________                                    

(6) An approximate chemical formula based on the elemental analysis ofC₄₅ H₈₀ O₂₂ N₁₀ S₂ with an approximate molecular weight of
 1200. 3. Aprocess for producing antibiotic A26201-1 as defined in claim 1 orantibiotic A26201-2 as defined in claim 2 which comprises cultivatingunder aerobic conditions Actinoplanes species A26201 or a mutant thereofin a suitable culture medium containing a carbon source and a nitrogensource at a pH of about 6 to 8 and at a temperature of about 20° C. to35° C. until a sufficient amount of either or both of the antibioticsare produced by said microorganism in said culture medium.
 4. Theprocess of claim 3 wherein said culture medium includes one of the groupconsisting essentially of fructose, sucrose, tryptophan or valine.
 5. Aprocess for inhibiting bacteria which comprises applying to saidbacteria or habitat thereof an effective antibacterial amount ofantibiotic A26201-1 as defined in claim 1 or antibiotic A26201-2 asdefined in claim
 2. 6. The process of claim 5 wherein said bacteria aregram positive bacteria.
 7. A process for promoting the growth of animalswhich comprises administering to said animals an effective growthpromoting amount of antibiotic A26201-1 as defined in claim 1 orantibiotic A26201-2 as defined in claim
 2. 8. The process of claim 7wherein said animals are monogastric animals.
 9. The process of claim 7wherein said animals are ruminant animals.
 10. An animal feedcomposition for promoting the growth of animals which comprises amixture of standard animal feed and an effective growth promoting amountof antibiotic A26201-1 as defined in claim 1 or antibiotic A26201-2 asdefined in claim
 2. 11. The composition of claim 10 wherein saidstandard animal feed is standard animal feed for monogastric animals.12. The composition of claim 10 wherein said standard animal feed isstandard animal feed for ruminant animals.
 13. The animal feedcomposition of claim 11 wherein the concentration of said antibiotic orantibiotics is about 5 to about 25 parts per million of the ultimatefeed composition.
 14. The animal feed composition of claim 12 whereinthe concentration of said antibiotic or antibiotics is about 2 to about25 parts per million of the ultimate feed composition.
 15. A process forobtaining separate substances, antibiotic A26201-1 as defined in claim1, and antibiotic A26201-2 as defined in claim 2 which comprisescultivating under aerobic conditions Actinoplanes species A26201 (ATCC39573) in a suitable culture medium containing a carbon source and anitrogen source at a pH of about 6 to 8 and at a temperature of about20° C. to 35° C. until a sufficient amount of either or both of theantibiotics are produced by said microorganism in said culture medium,recovering the crude fermentation broth containing antibiotics,partially purifying the antibiotics by use of a preparative reversephase liquid chromatography system, and resolving the two antibiotics byuse of a semi-preparative high performance liquid chromatography system.